Allergenicity and structural properties of new Cor a 1 isoallergens from hazel identified in different plant tissues

The hazel allergen Cor a 1 is a PR-10 protein, closely related to the major birch pollen allergen Bet v 1. Hazel allergies are caused by cross-reactive IgE antibodies originally directed against Bet v 1. Despite the importance of PR-10 proteins in allergy development, their function and localization in the plant remain largely elusive. Therefore, the presence of Cor a 1 mRNA and proteins was investigated in different tissues, i.e., the female flower, immature and mature nuts, catkins, and pollen. Four yet unknown Cor a 1 isoallergens, i.e., Cor a 1.0501–1.0801, and one new Cor a 1.03 variant were discovered and characterized. Depending on the isoallergen, the occurrence and level of mRNA expression varied in different tissues, suggesting different functions. Interestingly, Cor a 1.04 previously thought to be only present in nuts, was also detected in catkins and pollen. The corresponding Cor a 1 genes were expressed in Escherichia coli. The purified proteins were analysed by CD and NMR spectroscopy. Immunoblots and ELISAs to determine their allergenic potential showed that the new proteins reacted positively with sera from patients allergic to birch, hazel and elder pollen and were recognized as novel isoallergens/variants by the WHO/IUIS Allergen Nomenclature Sub-Committee.

There appear to be more Cor a 1 isoallergens and variants present in hazel than have been described so far, as indicated by similarities of genes found in the hazel genome 32 .Although the presence of the isoallergens Cor a 1.01 and Cor a 1.04 in pollen and kernel, respectively, has been shown, the expression and localization of the other known isoallergens Cor a 1.02 and Cor a 1.03 in different tissues has not yet been clarified.
To shed more light on the occurrence and distribution of Cor a 1 isoallergens, we determined the presence of Cor a 1 mRNA in different hazel plant tissues and in pollen.Since the fully assembled and annotated genome sequence of a European hazel species, C. avellana cv 'Tombul' was recently published 32 it was possible to search specifically for novel Cor a 1 genes and their expression in different hazel tissues.
Total RNA was isolated from the female flower, immature and mature nuts, male catkins, and pollen.After cDNA production with an mRNA-specific poly(T) primer, PCR was performed using specific primers binding either to the 5′ or 3′ end of the gene (only Cor a 1.0302) or to the 5′ and 3′ untranslated regions (UTRs).Since the UTR regions of variants of a certain isoallergen are rather similar or even identical 32 , it was not possible to distinguish different variants using this method.Thus, only the existence of a specific isoallergen, possibly consisting of several variants, could be determined in a specific plant material (Figs. 1, S6).However, this procedure allowed us to use the PCR sample for cloning and for sequencing the complete amplified gene for the identification of new isoallergens (Table S1).To verify that the amplificates contained Cor a 1 DNA, all PCR probes were subjected to DNA sequencing.To detect even extremely low specific mRNA levels, a 2nd PCR amplification step was carried out in such cases with the gel purified DNA of the first PCR.
So far, the Cor a 1.01 protein has been detected in hazel pollen 29 .However, the mRNA expression analyses presented here indicate that Cor a 1.01 is, in addition, expressed in minimal amounts in the female flower and in the mature nut as indicated by the 2nd PCR amplification step.Significantly stronger specific mRNA levels could be detected in the immature nut as well as in catkins and pollen.
Cor a 1.02 mRNA was present in the female flower and the mature nut, and high mRNA concentrations were visible in the immature nut, but no specific mRNA was discovered in the male tissues.Moreover, a new, putative Cor a 1.02 variant, designated Cor a 1.02-Cav01g11820 was detected via sequencing of the PCR product (Figure S1).However, since it was not possible to purify the protein from E. coli to determine its allergenic properties, this variant was not considered further in our studies.
Cor a 1.03 mRNA was present in all tissues with the exception of the mature nut even though high mRNA levels were detected in the immature nut.Although we could detect a very faint band after the first PCR round of the mature nut, sequencing of the amplificate of the 2 nd PCR revealed no Cor a 1.03 gene sequence (Fig. 1).In addition, using LC-MS E (liquid chromatography-mass spectrometry elevated energy ) variants of Cor a 1.01 and Cor a 1.03 could also be discovered on the protein level by LC-MS E in pollen extract (Figure S2).
It has been shown previously that Cor a 1.04 is present in nuts 30 .Since Cor a 1.04 was found to bind to a ligand isolated from pollen extracts it was suggested that it might play a role in fertilization 17 .Thus, it was interesting to determine whether Cor a 1.04 is localized in other tissues as well.Surprisingly, the 2nd PCR amplification step Figure 1.Expression of Cor a 1 genes in different hazel tissues.The RT-PCR products using gene specific primers to determine Cor a 1 mRNA expression were loaded on 1.5% agarose gels.In case no band was visible, a gel slice at the corresponding height was cut out.The purified mRNA was then used for the 2nd PCR.F: female flower; iN: immature nut; N: mature nut; C: catkin; P: pollen; ((+)) very low, (+): low, +: moderate, ++: strong expression levels.The RT-PCR samples used for cloning of the genes into expression vectors are marked with *M: 100 bp DNA standard (New England Biolabs; Frankfurt, Germany).18S rRNA isolated in a similar fashion was used as a control.
followed by sequencing revealed the presence of Cor a 1.04 specific mRNA also in catkins and pollen, albeit at very low amounts (Fig. 1).Thus, we show for the first time that Cor a 1.04 expression also occurs in the male tissues.This result provides a reasonable explanation why a ligand isolated from pollen extracts bound specifically to Cor a 1.0401 which was thought to be only present in the nut.
In addition, Cor a 1.04 mRNA could also be detected in the female flower and the immature nut (Fig. 1).Furthermore, LC-MS E data showed the presence of the Cor a 1.04 protein in extracts of the mature nut (Figure S2).However, only a few peptides could be confirmed, suggesting that protein isolation from nuts might be less efficient than mRNA extraction.In addition, no amplification step is performed before the MS analysis.This might also explain why other isoallergens (Fig. 1) could not be identified at the protein level by LC-MS E .Moreover, it is also possible that protein amounts in nuts are lower and do not necessarily correspond to mRNA levels.
Cor a 1.05 mRNA was found in the female flower and in the immature nut, however, in the mature nut, as well as in catkins and pollen specific mRNA was only visible after the 2nd PCR procedure (Fig. 1).
Cor a 1.06 mRNA was detected in the female flower, as well as in the immature and mature nut, and was also visible in pollen after the 2nd PCR.Cor a 1.07 mRNA levels were very low in all tissues.In contrast, Cor a 1.08 mRNA concentration was high only in the immature nut, whereas it was weak in the female flower and mature nut, and no expression could be verified in the male tissues (Fig. 1).
It is important to note, that our intention was to identify new expressed Cor a 1 genes by detecting the corresponding mRNAs in different tissues and to clone the genes.Our data represent only one specific time point, which means that Cor a 1 isoallergens that were not visible in our experiments might nevertheless be present at an earlier or later stage of development.
The observation that the mRNA expression of the individual isoallergens differs so strongly implies different properties and functions in the plant, e.g. during fertilization and kernel development, that still need to be explored.Different roles of Cor a 1 isoallergens have already been suggested as only the Cor a 1.04 isoallergen but not Cor a 1.01 binds the flavonoid derivative Q3O-(Glc)-Gal 17 .In summary, the data show that there are more Cor a 1 isoallergens than previously known and that their presence in mature nuts and pollen could play a role in hazelnut allergy (see below).

Identification of additional variants
In order to identify new isoallergens and variants, the amplified DNA products from specific tissues were cloned into the pCR-Blunt vector (Thermo Fisher, Schwerte, Germany) and sequenced.It turned out that several highly similar variants of a certain isoallergen could be detected in the different tissues tested (Figures S1, and S3).For some of them, only gene fragments could be amplified (data not shown).Due to the high similarity of the variants only one variant per isoallergen was selected and included in our studies to verify the biochemical and biophysical properties as well as the allergenicity (Figure S1).Furthermore, we identified variants of several isoallergens that could not be detected in the genome of the Turkish cultivar "Tombul" 32 by BLAST analyses, indicating genetic differences of C. avellana used in our studies.These genes are labelled with a GenBank accession number only, whereas a Cav01 number represents genes also found in the "Tombul cultivar".Our data indicate that for each isoallergen additional potential variants are present (Figure S3).However, due to their high similarity we did not include different variants of a certain isoallergen in our study.
As described above, we were able to identify four new isoallergens, namely Cor a 1.0501, Cor a 1.0601, Cor a 1.0701 and Cor a 1.0801 on the mRNA level (Figure S1).In addition to those isoallergens a new Cor a 1.03 variant, designated Cor a 1.0302 was detected.These proteins were included in biophysical and immunological studies.Due to their allergenic potential (see below), the four new isoallergens were recognized by the WHO/ IUIS Allergen Nomenclature Sub-Committee and named as described.
Amino acid sequence alignments and the identity matrix of Cor a 1 isoallergens and variants revealed that the new proteins possess sequence identities to the already known isoallergens of 46.88 -84.38%.The new Cor a 1.0302 variant is 96.23% identical to Cor a 1.0301 (Table 1).

Biochemical and biophysical analyses
The identified genes were cloned into the expression vectors pETGB1a or pET11a (only Cor a 1.0302) and expressed in E. coli (Table S2).The SDS polyacrylamide gel of the purified proteins exhibits apparent molecular masses of ca.16 kDa to 18 kDa (Figure S4).
To determine the refolding capacity and stability of the proteins, CD spectra were recorded (Fig. 2, panel  A, B).At 25 °C the spectra of the recombinant (r)Cor a 1 isoallergens are characteristic for proteins harboring α-and β-like secondary structures (Fig. 2, panel A).After heating to 95 °C, rCor a 1.0302, 1.0501, 1.0601, and 1.0701 unfolded as indicated by the minimum at 200 nm.In contrast, rCor a 1.0801 appeared to be more stable.The curve after heating to 95 °C (Fig. 2, panel A, red curve) changed only slightly and there was no discrete minimum at 200 nm, signifying that only a few sections of the protein became unfolded.After cooling to 25 °C, rCor a 1.0302, 1.0501, 1.0601 and 1,0701 almost completely refolded whereas rCor a 1.0801 was still folded and the proportion of unfolded protein moieties decreased as compared to the spectrum at 95 °C (Fig. 2, panel A, green curves).
rCor a 1.0302, 1.0601 and 1.0701 revealed relatively similar T m -values of 68.29, 54.08 or 62.68 °C.Cor a 1.0501 exhibited the highest T m -value of 84 °C (Fig. 2, panel B).rCor a 1.0801 displayed a biphasic melting behaviour during heating to 95 °C.However, complete denaturation could not be achieved.Due to the small differences of the denaturation and renaturation curves, the noise in the measurement of the melting curve of Cor a 1.0801 is quite high (Fig. 2, panel B).
Cor a 1.0801 shows a lower sequence identity to Bet v 1.0101 and also to most of the other Cor a 1 isoallergens (Table 1).This difference could be the reason for its higher thermostability.Previously, the structure of the www.nature.com/scientificreports/hypothetical protein TTHA0849 from the thermophilic bacterium Thermus thermophilus had been determined, which also shows the typical PR-10 fold of Bet v 1 33 .However, sequence comparisons revealed no evidence of conserved regions in Cor a 1.0801 and TTHA0849 that might explain its thermostability.Despite the similar three-dimensional structures, the sequence identity between Cor a 1.08 and TTHA0849 is only 20.98%, which is even lower than that of all the other Cor a 1 isoallergens to TTHA0849, except Cor a 1.05 (20.42%) (Figure S7).While CD spectra can be used to determine secondary structures, 1D NMR spectroscopy can reveal threedimensional folding of a protein.The 1D NMR spectra showed dispersion of the amide proton signals and high-field shifted signals of the methyl groups, which confirmed a three-dimensional folding for all isoallergens (Fig. 2, panel C).
These data suggest that the high stability and refolding capacity of the proteins could have an impact on their allergenicity.Heating might not completely abolish the allergenicity in hazel nut containing food stuff as Cor a 1 proteins could refold if the food is cooled down.

IgE binding
The rCor a 1 proteins purified from E. coli were further used for immunological studies in order to determine their allergenicity.Sera from 20 tree-pollen allergic individuals that had been tested positive for specific IgE (Spez.IgE Kit from Gold Standard Diagnostics, Kassel, Germany) against a mixture of birch, hazel and alder pollen were used for immunoblot analyses with the recombinant isoallergens.Only sera reaching values ≥ 3.5 U/ml corresponding to the enzyme-allergo-sorbent-test (EAST) class 3 or higher were selected.As expected, all sera reacted strongly with the Bet v 1 control from birch since this is usually the sensitizing agent 4 (Fig. 3, Figure S5).Furthermore, all sera reacted with rCor a 1.0101, albeit with varying intensities.Similarly, IgE binding could be detected with all sera employing rCor a 1.0302, 1.0401, 1.0501, and 1.0601, indicating that all of them are potent allergens.Using rCor a 1.0701, serum #1, #9, #10, and #13 displayed no IgE binding.rCor a 1.0801 proved to be the weakest allergen and showed IgE binding only with seven sera, namely #11, #12, #14, #16, #17, #18, and #20.Obviously, the sera responded better to those Cor a 1 isoallergens which possess a high identity with the sensitizing Bet v 1.0101 protein (Table 1).The weak allergens Cor a 1.0701 and Cor a 1.0801 have only 54.38 and 46.88% identity with Bet v 1.0101, respectively.
Since we have previously described that denaturation of an allergen during the blotting procedure can impair IgE binding 34 , ELISAs were chosen as an additional method to confirm the allergenicity of the isoallergens (Table 2).Here, all sera reacted with rCor a 1.0101 to rCor a 1.0601.With the exception of serum #12, all sera appeared to be less reactive using Cor a 1.0701.However, in contrast to the Western Blots, rCor a 1.0701 exhibited  IgE binding with all sera, indicating that denaturation of the antigen in the Blots could play a role.Furthermore, the ELISA might be more sensitive than the Blot.Interestingly serum #12 was highly reactive with all isoallergens other than Cor a 1.0801.In addition to the sera reacting with Cor a 1.0801 in the above Western Blot, Cor a 1.0801 also showed weak IgE binding using serum #19.These results confirm that Cor a 1.0801 is a weaker allergen than the others but nevertheless several individuals appear to possess reactive IgE antibodies.

Concluding remarks
Our data show that there exist more isoallergens and variants of Cor a 1 than previously known, e.g., four potential variants of the newly identified isoallergen Cor a 1.05 as well as three additional variants of Cor a 1.03 could be detected (Figs. 4, S1, S3).We have already shown that Cor a 1.0401 binds to the specific natural ligand Q3O-(Glc)-Gal 17 .To obtain further information on ligand binding, we are currently conducting experiments to show whether the newly identified isoallergens have different ligand binding affinities.Since little was previously known about the expression of Cor a 1 in different plant tissues the results presented here (Fig. 1, Table 1) show for the first time, that the expression of the isoallergens studied is dependent on the plant tissue, e.g.Cor a 1.01 could be found in all tissues, whereas Cor a 1.08 was only present in the female tissues, albeit at different mRNA expression levels.Obviously, the localization of a certain isoallergen has an impact on its expression level (Fig. 1).These differences suggest different functions of the isoallergens, e.g., during plant development, fertilization and germination, that remain to be elucidated in the future.
The mRNA levels of Cor a 1.01 and Cor a 1.03 are high in pollen (Fig. 1) and thus the proteins appear to play an important role as triggers of pollen allergy.In contrast, the other isoallergens, present in mature nuts, might be relevant for allergic reactions caused after intake of hazel nut containing foodstuff (Fig. 1).

Figure 3.
Immunoblots.SDS-PAGE (16% PA gels) was performed with the Cor a 1 proteins, followed by blotting onto a 0.2 µm nitrocellulose membrane as indicated on top of the immunoblots.1.5 µg/cm protein was blotted except for Cor a 1.0801 with 0.8 µg/cm.The membranes were cut into stripes and incubated with patients' sera.Bound specific IgE antibodies were detected using a mouse-anti-human IgE antibody coupled to alkaline phosphatase followed by nitroblue tetrazoliumchloride (NBT)/5-Bromo-4-chloro-3-indolylphosphate (BCIP) staining.The grey arrows indicate weak IgE binding for nCor a 1 from hazel pollen and hazel nut extract and for rCor a 1.0801.Labels on top of the immunoblots: A: secondary antibody control; N: horse serum; 1-20: sera of patients allergic against tree pollen.
The allergenic potential of Cor a 1 proteins is likely to be enhanced by the folding stability of the isoallergens (Fig. 2).All isoallergens regained their secondary structure elements after short heating to 95 °C and recooling (Fig. 2, panel A).Similar results have already been shown for Bet v 1.0101 as well as for the homologous carrot allergen Dau c 1 (Jacob u. a. 2020; Machado u. a. 2016; Hendrich u. a. 2023).For Bet v 1.0101 it has been demonstrated that the increased fold stability of the protein has an impact on its allergenicity 36 .The Dau c 1.0105 Table 2. ELISA with purified rCor a 1 isoallergens and sera of tree pollen allergic patients.The values represent the ratio of the measurements at 450/620 nm normalized to the 450/620 ratio of the negative control (horse serum).Serum dilution 1:10.Two technical replicates were performed.Light yellow: 1-1.5, yellow: 1.5-5, orange: 5-10, red: 10-20, purple: > 20.Europe GmbH, Freiburg, Germany) by a stepwise increase (15%, 25%, 50%, 70%, 100%) of the imidazole concentration.

CD und NMR spectroscopy
Circular dichroism (CD) spectra and standard 1D 1 H NMR spectra were recorded as previously described 37 .

Patients' Sera
Sera were obtained from patients after informed consent and approval by the Ethics Committee of the University of Bayreuth.All experiments were performed in accordance with relevant guidelines and regulations.Sera from patients diagnosed with seasonal (early spring) rhinitis, cough and breathing difficulties were tested for specific IgE against a tree pollen mixture including birch, hazel and alder pollen (Spez.IgE Kit from Gold Standard Diagnostics, Kassel, Germany).Only sera reaching values ≥ 3.5 U/ml corresponding to the enzyme-allergo-sorbenttest (EAST) class 3 or higher were selected and tested further against Bet v 1.0101 (used as a control), as well as hazel pollen extract and an extract from mature hazel nuts to confirm the presence of specific IgE antibodies against Cor a 1 proteins.Those sera were used in this study to detect new Cor a 1 isoallergens.

IgE immunoblots and IgE ELISAs
IgE immunoblots and ELISAs were performed similarly to previously described procedures 37,39 .Mouse antihuman IgE coupled to alkaline phosphatase (BD Bioscience, Heidelberg, Germany) and AP Conjugate Substrate Kit (Bio-Rad, Germany) was used for immunoblots.Horse serum was used as a negative control and a commercially available mixture of sera from birch pollen and hazel pollen allergic patients (Gold Standard Diagnostics Kassel GmbH) as a positive control.
For ELISAs 2 µg/ml antigen (Cor a 1 proteins or Bet v 1.0101) was coated on high binding ELISA plates F (Sarstedt, Nümbrecht, Germany) in coating buffer (50 mM Na 2 CO 3 /NaHCO 3 pH 9.6) overnight at 4 °C.After washing three times with PBS-T (phosphate buffered saline, 0.05% Tween 20) and blocking with blocking buffer (PBS-T containing 2% BSA) for 1 h at room temperature (RT), sera (diluted 1:10 with blocking buffer) were added and incubated overnight at RT.After washing four times with PBS-T, bound specific IgE was detected with horse radish peroxidase (HRP) labeled mouse-anti-human IgE-Fc-HRP (SouthernBiotech, Birmingham, USA) (diluted 1:5000 with blocking buffer).After 2 h at RT and washing with PBS-T, 100 µl substrate solution of the KPL TMB Microwell Peroxidase Substrate System (2-C) (Sera care Life Science, MA, USA) was added.The color reaction was stopped with 25% sulfuric acid (50 µl/well) and absorbance was read at 450 nm and 620 nm as a reference.

Figure 4 .
Figure 4. Amino acid sequence alignment of identified cor a 1 genes expressed in Corylus avellana.The newly identified isollargens are highlighted in green and are shown in comparison with the known Cor a 1 sequences.* conserved amino acids; : conservative exchanges; .semi-conservative exchanges.

Table 1 .
Identity matrix of Cor a 1 isoallergens and variants.Numbers indicate the amino acid sequence identity in %